Biomedical research has made rapid progress based on sequential processing of biological samples. Sequential processing techniques have resulted in important discoveries in a variety of biologically related fields, including, among others, genetics, biochemistry, immunology and enzymology. Historically, sequential processing involved the study of one or two biologically relevant molecules at the same time. These original sequential processing methods, however, were quite slow and tedious. Study of the required number of samples (up to tens of thousands) was time consuming and costly.
A breakthrough in the sequential processing of biological specimens occurred with the development of techniques of parallel processing of the biological specimens, using fluorescent marking. A plurality of samples are arranged in arrays, referred to herein as microarrays, of rows and columns into a field, on a substrate slide or similar member. The specimens on the slide are then biochemically processed in parallel. The specimen molecules are fluorescently marked as a result of interaction between the specimen molecule and other biological material. Such techniques enable the processing of a large number of specimens very quickly.
Some applications for imaging require two apparently contradictory attributes: high-resolution and high-content. The resolution requirement is driven by the need to have detail in the image that exceeds by at least 2× the information content of the object being images (the so called Nyquist Limit). The content requirement is driven by the need to have information over a large area. One method that addresses these needs is to acquire a plurality of individual images with high spatial resolution (panels) and to collect these panels over adjacent areas so as to encompass the large desired area. The multiple panels can then be assembled into a single large image based on the relative location of the optics and the sample when each panel was collected. When assembling the plurality of panels into a single montage, a number of steps may be taken to correct for intensity non-uniformities within each panel (known herein as flat-field Calibration and Panel Flattening) as well as non-uniformities in the panel to panel intensities.